Protein homologue of human angiogenin

ABSTRACT

The invention concerns a new protein of approximately 17 KD, with angiogenic activity, a process for isolating it from mammalian milk, therapeutic compositions containing it, a process for detecting and/or determining the content of mammalian angiogenins, their homologues and their fragments. Said protein, of bovine origin, has a sequence of 125 aminoacids, 81 of which are common to human angiogenin, and a molecular weight of approximately 17 KD, and is extracted from mammalian milk. Application to the detection of mammalian angiogenin.

The present invention relates to a novel protein of about 17 KD, withangiogenic action, to the method of isolating it from mammalian milk, totherapeutic compositions in which it is present, to a method ofdetection and/or determination and to immunological reagents fordetecting and/or determining mammalian angiogenins, homologs thereof andfragments thereof.

Following the pioneering work of J. FOLKMAN (q.v. J. Exp. Med., 133, 275(1971)), who demostrated that the growth of a tumor requires asubstantial blood supply, this being provided by the continuous growthof new blood vessels, he suggested that this growth results from thepresence of a diffusible substance which he called "Tumor AngiogenesisFactor" (TAF). Several proteins which stimulate angiogenesis wereisolated (FOLKMAN et al., Science, (1987), 235, 442). Among thesesubstances, angiogenin, a protein of human origin, was purified byVALLEE's team and its gene was cloned. In 1985, J. W. FETT et al.(Biochem., (1985), 24, 5480-5486) isolated human angiogenin fromcancerous human intestinal cells in culture. The intestinal cells HT 29secrete human angiogenin in the culture medium. 0.5 μg/liter of humanangiogenin was isolated from these serum-free culture media. VALLEE'steam determined the concentration of angiogenin first by Bradford'smethod (Anal. Biochem., 1975, 72, 248-254, a stain fixing method usingSAB as the standard) and then by the method described in Biochem., 1986,25, 3527-3732, by SHAPIRO et al. The amino acid sequence was alsospecified by STRYDOM et al. (BIOCHEMISTRY, (1985), 24, p. 5486-5494).

Human angiogenin is a protein with a molecular weight of 14,400 D.

The angiogenin isolated from human tumoral cells consists of a singleprotein chain comprising 123 amino acids and having the followingsequence: ##STR1##

The C-terminal amino acid is proline; three disulfide bridges link thecysteines 26-81, 39-92 and 57-107.

The sequence of human angiogenin is 35% homologous with that of humanpancreatic ribonuclease, in particular with regard to the amino acidsessential for ribonucleolytic activity (q.v. BIOCHEMISTRY, (1985), 24,5494-5499, KURACHI et al.). The activity of human angiogenin isconsiderable since 50 ng, i.e. 3.5 picomoles, are capable of causingvascularization of the rabbit cornea and 35 femtomoles are capable ofinducing vascularization of the chicken embryo.

It is known to prepare human angiogenin by cloning. Cloning of the genecoding for human angiogenin makes it possible to prepare satisfactoryamounts via suitable expression systems. However, such methods areexpensive.

It has proved necessary to look for a compound which has propertiessimilar to those of human angiogenin and whose method of preparation issimpler and less expensive.

One object of the present invention is consequently to provide a novelprotein which has properties similar to those of human angiogenin and isobtained by inexpensive means which are easy to carry out and affordhigh quantitative yields.

Another object of the invention is to provide a novel method ofobtaining the said protein which does not have the disadvantages of themethods of the prior art; in fact, this novel method makes it possibleto obtain large amounts of the protein in question at very low cost,which has important advantages in the context of the industrialmanufacture of pharmaceutical compositions containing this protein.

Yet another object of the invention is to provide pharmaceuticalcompositions containing the said protein.

Another object of the invention is to provide an agent for detecting anddetermining mammalian angiogenins, homologs thereof and fragmentsthereof in biological fluids.

Another object of the invention is to provide a kit for detecting anddetermining the above-mentioned proteins in the said fluids.

The present invention relates to a protein which has a sequencecomprising 125 amino acids and has formula I below: ##STR2## whosesequence has 81 amino acids in common with human angiogenin and whosemolecular weight is about 17 KD.

The molecular weight was evaluated by comparing the electrophoreticmigration speed of the said bovine protein with that of the followingreferences: myoglobin (MW: 17,200), myoglobin 1+2 (MW: 14,600),myoglobin A (MW: 8240), myoglobin 2 (MW: 6380) and myoglobin 3 (MW:2560) (Pharmacia).

In the composition of the said 17 KD protein, determined after totalacid hydrolysis, the following amino acids are present in theproportions given: Phe: 6, Leu: 4, Ile: 9, Met: 2, Val: 4, Pro: 7, Ser:6, Thr: 6, Ala: 4, Tyr: 6, His: 6, Glu(Gln): 10, Asp(Asn): 16, Lys: 9,Arg: 15, Gly: 9, Cys: 6.

According to the invention, the said protein is obtained by extractionfrom mammalian milk, especially cow's milk, by cloning or by a syntheticmethod.

The present invention further relates to peptides which constitutefragments of the 17 KD protein according to the invention; it covers inparticular:

- a peptide having the following amino acid sequence:

Glu-Asp⁶⁰ -Arg-Asn-Gly-Gln-Pro⁶⁵ -Tyr-Arg-Gly-Asp-Leu⁷⁰ -Arg-Ile-Ser,

in which 9 out of 15 residues are aligned with the sequence 58-72 ofhuman angiogenin;

- a peptide having the following amino acid sequence:

Phe-Asp-Glu-Ser-Phe¹²⁰ -Ile-Thr-Pro-Arg-His¹²⁵,

which corresponds to the C-terminal fragment of the 17 KD protein;

- a peptide having the following amino acid sequence:

Glu-Asn¹¹⁰ -Gly-Leu-Pro-Val-His¹¹⁵ -Phe, which is aligned with thesequence 108-115 of human angiogenin, in which sequence 7 out of 8residues are identical;

- a peptide having the following amino acid sequence:

Ile-Val¹⁰⁵ -Val-Gly-Cys-Glu,

in which 4 out of 6 residues are aligned with the sequence 103-108 ofhuman angiogenin;

- a peptide having the following amino acid sequence:

Arg-Tyr-Ile-His-Phe¹⁰ -Leu-Thr-Gln-His-Tyr¹⁵ -Asp-Ala-Lys,

in which 11 out of 13 residues are aligned with the sequence 5-17 ofhuman angiogenin;

- a peptide having the following amino acid sequence:

Asn-Thr⁴⁵ -Phe-Ile-His-Gly-Asn⁵⁰ -Lys,

which is distinguished by total homology with the sequence 43-50 ofhuman angiogenin;

- a peptide having the following amino acid sequence:

Ile-Lys⁵⁵ -Ala-Ile-Cys-Glu,

which is also distinguished by total homology with the sequence 53-58 ofhuman angiogenin;

- a peptide having the following amino acid sequence:

Leu⁷⁰ -Arg-Ile-Ser-Lys-Ser⁷⁵ -Glu-Phe-Gln,

in which 8 out of 10 residues are aligned with the sequence 69-77 ofhuman angiogenin; and

- a peptide having the following amino acid sequence:

Arg⁶⁷ -Gly-Asp,

the said peptide being recognized by an endothelial cell receptor.

The present invention further relates to a method of obtaining the saidprotein according to the invention, wherein the said protein isextracted from mammalian milk, especially cow's milk.

In an advantageous way of carrying out the method according to theinvention, the said protein is extracted by cation exchangechromatography followed by elution with an appropriate eluent.

According to an advantageous provision of this way of carrying out themethod, the eluent is an alkali metal salt of a weak organic acid,especially sodium acetate.

According to another advantageous provision, the eluted fraction issubjected to cation exchange chromatography a second time.

The protein isolated in this manner is purified by chromatography on agel filtration column.

The protein purified in this manner is obtained with a yield of theorder of 0.5 mg/liter of milk.

As a variant of this way of carrying out the method, the milk of bovineorigin is subjected to delipidation prior to extraction.

According to an advantageous provision, delipidation is effected bycentrifugation.

In an advantageous modification of this provision, centrifugation iscarried out at 4000 g for 30 min at a temperature of 4° C.

The present invention further relates to a therapeutic composition whichcomprises, as the active compound, the 17 KD protein and/or fragments orhomologs thereof, especially for the treatment of disorders whichrequire the growth of blood vessels to be inhibited or increased.

The therapeutic compositions according to the invention can be used inall pathologies in which there is a problem of vascularization, andespecially wounds, scabs, ulcers, grafts and circulatoryinsufficiencies. They can also be used in cosmetology (skin, scalp).They can also be used in the field of veterinary medicine, especially inthe diagnosis of mastitis and the selection of milking cows.

The present invention further relates to an immunological reagent fordetecting or determining mammalian angiogenins which is selected fromthe group comprising antibodies directed against the 17 KD protein andantipeptide antibodies, especially antibodies directed against one ofthe peptides defined above, the said antibodies being used by themselvesor in a mixture.

The present invention further relates to a method of detecting anddetermining mammalian angiogenins in biological fluids, wherein ananti-angiogenin antibody, especially an antibody directed against the 17KD protein or an anti-peptide antibody, according to the invention, isreacted, under appropriate conditions, with a biological fluid thoughtto contain the said angiogenin, the reaction being evaluated by anappropriate means such as, in particular, RIA, ELISA orimmunofluorescence.

The present invention further relates to a kit for detecting and/ordetermining mammalian angiogenin, and especially human angiogenin, inbiological fluids, which comprises:

an appropriate amount, subdivided into unit doses if necessary, ofanti-angiogenin antibodies, in particular antibodies directed againstthe 17 KD protein or antipeptides antibodies, especially antibodiesdirected against one of the peptides defined above; and

if necessary, an appropriate amount of buffers, diluents and reagentsrequired to carry out the said detection and/or determination.

In addition to the foregoing provisions, the invention also includesother provisions which will become apparent from the followingdescription referring to an Example of the preparation of the proteinaccording to the invention and to an account of experiments performed:

to demonstrate the homology between human angiogenin and the 17 KDprotein of bovine origin, according to the invention;

to demonstrate the homology with ribonuclease; and

to demonstrate the activity of the said 17 KD protein of bovine originon angiogenesis.

It must be clearly understood, however, that these Examples and thisaccount of experiments are given solely in order to illustrate thesubject of the invention, without in any way implying a limitation.

"Sephadex"®, "Sepharose"®, "Biogel"® and "Phenyl Superose"® are wellknown trademarks in the field of chromatography. "Sephadex" and"Sepharose" are dextran polymers on which anionic or cationic functionshave been grafted. "Phenyl Superose"® corresponds to cross-linkedagarose. "Biogel P gels" are porous polyacrylamide beads prepared bycopolymerization of acrylamide and N,N'-methylene-bis-acrylamide.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the diagram of the elution of the 17 KD protein from thePhenyl Superose column, the time in minutes being plotted on theabscissa and the optical densities on the ordinate. Peak 2 contains thepure 17 KD protein.

EXAMPLE A: Extraction of the 17 KD Protein from Cow's Milk

50 liters of cow's milk are delipidated by centrifugation (4000×g-30min-4° C.) and chromatographed on a column of SP-Sephadex C50 (10×100cm) (Pharmacia).

The column is washed with 10 l of a 0.35M solution of sodium acetate, pH7, and then eluted with 5 l of 1.5M sodium acetate, pH 8. The solutionobtained is diluted to give a sodium acetate concentration of 0.2M. Thissolution is chromatographed on a column of S-Sepharose Fast Flow (10×100cm) (Pharmacia) which has been equilibrated beforehand with 0.22M sodiumacetate, pH 6.5. After the column has been washed with the same acetatesolution, the protein is eluted with one liter of a 0.4M solution ofsodium acetate.

Salt is removed from this solution by gel filtration chromatography on acolumn of Bio-gel P-30 (4×100 cm).

After the column has been eluted with water, the fraction containing theprotein in chromatographed on a column of Phenyl Superose HR5/5(Pharmacia) by FPLC (Fast Protein Liquid Chromatography). Elution isperformed using a gradient obtained by mixing buffer A-50 mM sodiumphosphate, 1.7M ammonium sulfate, pH 7-with buffer B-50 mM sodiumphosphate, pH 7 (time 0 minute: 100% buffer A; time 15 minutes: 100%buffer A; time 40 minutes: 0% buffer A).

FIG. 1 attached shows the diagram of the elution of the 17 KD proteinfrom the Phenyl Superose column, the time in minutes being plotted onthe abscissa and the optical densities on the ordinate. Peak 2 containsthe pure 17 KD protein. The yield is 25 mg of protein, i.e. 0.5 mg/l ofdelipidated milk.

TESTS EXAMPLE 1: Homology Between Human Angiogenin and the 17 KD Protein

The data which we have available on the 17 KD protein show itsrelationship with human angiogenin; in fact, 81 amino acids are commonto both proteins.

Amino acid analysis, evaluated for human angiogenin and determined aftertotal acid hydrolysis for the 17 KD protein of bovine origin (Table Ibelow), shows that the two proteins have very similar compositions.

                  TABLE I                                                         ______________________________________                                        COMPARATIVE AMINO ACID ANALYSIS OF HUMAN                                      ANGIOGENIN AND THE 17 KD PROTEIN OF BOVINE                                    ORIGIN                                                                                      HU    BO                                                        ______________________________________                                        Phe             5       6                                                     Leu             6       4                                                     Ile             7       9                                                     Met             1       2                                                     Val             5       4                                                     Pro             8       7                                                     Ser             9       6                                                     Thr             8       6                                                     Ala             5       4                                                     Tyr             3       6                                                     His             6       6                                                     Glu(Gln)        10      10                                                    Asp(Asn)        14      16                                                    Lys             7       9                                                     Cys             6       6                                                     Trp             1       --                                                    Arg             14      15                                                    Gly             8       9                                                     Total*          116     119                                                   ______________________________________                                         *without Cys and Trp                                                     

The comparison of the sequences of human angiogenin and the 17 KDprotein of bovine origin which is given in Table II below shows ahomology of more than 60%. Most of the differences are the result ofconservative substitutions.

The 6 cysteine residues are in the same position, which indicates atertiary structure similar to that of human angiogenin, a single shiftbeing necessary in the C-terminal region for alignment; only the N- andC-terminal regions are responsible for the difference in size betweenthe two proteins (the 17 KD protein has two amino acids more than humanangiogenin).

However, as distinct from human angiogenin, which has a pyroglutamicacid in the N-terminal position, the 17 KD protein of bovine origin hasan additional alanine in the N-terminal position.

This difference is explained by the presence of a signal peptide of 24(or 22) amino acids (J. FETT et al., Biochem., (1985), 24, 5480),revealed by sequencing of the cDNA's coding for human angiogenin;suppression of this peptide takes place on treatment with the signalpeptidase, which cleaves between the alanine and the glutamine in:Pro-Pro-Thr-Leu-Ala⁻¹ -Glu⁺¹ -Asp-Asn. . . , in human angiogenin,whereas the presence of the N-terminal alanine in the 17 KD protein ofbovine origin is due to displacement of the linkage cleaved by thesignal peptidase, thereby leaving an additional alanine in theN-terminal position and avoiding the modification to pyroglutamic acidwhich is observed in the case of human angiogenin.

The sequence of the 17 KD protein, as shown in Table II below, wasdetermined by the conventional protein sequencing techniques.

                                      TABLE II                                    __________________________________________________________________________    COMPARATIVE SEQUENCES OF THE 17 KD PROTEIN AND ANGIOGENIN                     __________________________________________________________________________     ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                     __________________________________________________________________________

EXAMPLE 2-Homology with RNase

R. SHAPIRO et al. (Biochem., (1986), 25, 3527-3532) showed that, inpractice, all the residues of the sequence of the active site of bovineRNase are preserved in human angiogenin.

Furthermore, the observation that a human ribonuclease inhibitorsuppresses both the angiogenic and the ribonucleolytic activity of humanangiogenin confirms that the homology of angiogenin with RNase isfunctionally significant.

The following results confirm these observations (Table III below):

The homology of the 17 KD protein of bovine origin with bovine RNase(39% of the residues being identical) is similar to that of humanangiogenin with human RNase (34% of the residues being identical).

Except for the Arg/Ile mutation in position 43 and two conservativesubstitutions (Asp-69 for Asn-68 and Phe-116 for Leu-115), all theresidues which are known to be involved in the ribonucleolytic activityare preserved, both in human angiogenin and in the 17 KD protein ofbovine origin, indicating a strong selection pressure for maintainingthis significant functional homology (Table III).

The substitution of Phe-116 of the 17 KD protein of bovine origin forLeu-115 of human angiogenin is of particular interest: it has beenshown, for example, that the replacement of Phe by Leu in RNase causesthe activity to drop by a factor of 10. Thus, subject to the RNaseactivity being directly involved in its biological activity, thepresence of Phe-116 in the 17 KD protein could be an indication of agreater activity than that of human angiogenin.

Based on its homology with pancreatic ribonuclease A, thethree-dimensional structure of human angiogenin was evaluated when thesites affected by the mutations are reincorporated into this structure;in this case, it can be observed that, with the exception of the Arg/Ilemutation in position 43, all the substitutions occurring in thethree-dimensional structure of the protein which are involved in itsribonucleolytic activity are conservative substitutions.

                                      TABLE III                                   __________________________________________________________________________    HOMOLOGIES BETWEEN HUMAN ANGIOGENIN AND THE 17 KD PROTEIN                     AND BETWEEN BOVINE AND HUMAN RNases                                           __________________________________________________________________________     ##STR9##                                                                                ##STR10##                                                                                             ##STR11##                                   ##STR12##                                                                               ##STR13##                                                                                                 ##STR14##                               ##STR15##                                                                               ##STR16##                                                                                               ##STR17##                                 ##STR18##                                                                               ##STR19##                                                                                       ##STR20##                                        __________________________________________________________________________

EXAMPLE 3-BIOLOGICAL PROPERTIES OF THE 17 KD PROTEIN OF BOVINE ORIGIN

-Chicken egg chorioallantoic membrane test:

This test of biological activity has been described by FETT et al.(Biochem., (1985), 24, 5480-5486). It consists in assessing thedevelopment of blood vessels in the chorioallantoic membrane of thechicken embryo after the application of increasing amounts, of the orderof 10 ng to 1 μg, of the 17 KD protein of bovine origin. Out of 80fertilized eggs which were investigated in the presence of the 17 KDprotein, the development of new blood vessels was observed in 62 eggsfor an amount of 50 ng of angiogenin. These results show that the 17 KDprotein according to the invention has an effect on organogenesis andthe growth of blood vessels.

The fertilized eggs are placed in an incubator at 38° C. in anatmosphere of 70% humidity.

After 3 days, an aperture is made in the shell and sealed with agas-permeable membrane.

On day 5, a disk of a membrane soaked with a solution of the 17 KDprotein is implanted on the chorioallantoic membrane.

The development of the blood vessels which converge towards the disksoaked with angiogenin is followed on days 8, 9 and 10 using binoculars.

The 17 KD protein of bovine origin according to the invention can beused in a pharmaceutically acceptable form for the treatment ofdisorders which require the growth of blood vessels in man to beinhibited or increased.

As is apparent from the foregoing description, the invention is in noway limited to those embodiments and methods of application which havenow been explicitly described; on the contrary, it encompasses all thevariants thereof which may occur to those skilled in the art, withoutdeviating from the framework or the scope of the present invention.

What is claimed is:
 1. An isolated and purified protein which has asequence comprising 125 amino acids and has formula I below: ##STR21##whose sequence has 81 amino acids in common with human angiogenin andwhose molecular weight is about 17 KD.
 2. A method of obtaining theprotein according to claim 1 comprising: extracting cow's milk andrecovering said protein.
 3. The method according to claim 2 wherein theproduct obtained is purified by chromatography on a gel filtrationcolumn.
 4. The method according to claim 2 wherein the milk is firstdelipidated by centrifugation.
 5. The method according to claim 2wherein extraction is carried out by cation exchange chromatographyfollowed by elution with an appropriate eluent.
 6. The method accordingto claim 5 wherein the product obtained is purified by chromatography ona gel filtration column.
 7. The method according to claim 5 wherein theeluent contains sodium acetate or another alkali metal salt of a weakorganic acid.
 8. The method according to claim 5 or 7 wherein the elutedfraction is subjected to cation exchange chromatography a second time,followed by elution with an appropriate eluent a second time.
 9. Themethod according to claim 8 wherein the product obtained is purified bychromatography on a gel filtration column.
 10. The method according toclaim 8 wherein the eluent contains sodium acetate or another alkalimetal salt of a weak organic acid.
 11. The method according to claim 10wherein the product obtained is purified by chromatography on a gelfiltration column.
 12. The method according to claim 7 wherein theproduct obtained is purified by chromatography on a gel filtrationcolumn.